Synergistic antiviral composition

ABSTRACT

Combinations of 9-(1,3-dihydroxy-2-propoxymethyl) guanine or a pharmaceutically acceptable salt thereof, with β-interferon show a surprisingly high degree of synergism in their activity against viral infections.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an antiviral composition comprising9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and β-interferon(β-IFN). This invention also relates to a method of treating viralinfections using the above composition or by co-administration of DHPGand β-interferon.

2. Related Disclosure

The compound 9-(1,3-dihydroxy-2-propoxymethyl)guanine is known to be apotent antiviral agent. See, for example, U.S. Pat. No. 4,355,032. It isknown to combine interferon with the antiviral compound acyclovir. SeeInfect.Immun. 32, 995-9 (1981), Antimicrob. Agents Chemother. 19, 672-4(1981) and Arch. Virol 73, 135-143 (1982).

It is known that the combinaton of DHPG and α-interferon and/orγ-interferon produces a synergistic effect and is a more potentantiviral agent than DHPG or interferon alone. See, e.g., U.S. Pat. No.4,462,986.

It has now feen found that the combination of DHPG with β-interferon issurprisingly more synergistic than the combination of DHPG with eitheror both α- or γ-interferon.

SUMMARY OF THE INVENTION

One aspect of the invention is an antiviral composition comprising9-(1,3-dihydroxy-2-propoxymethyl)guanine or a pharmaceuticallyacceptable salt thereof and β-interferon.

Another aspect of the invention is the method of treating viralinfections in a mammal by administering an effective amount ofβ-interferon and an effective amount of9-(1,3-dihydroxy-2-propoxymethyl)guanine or a pharmaceuticallyacceptable salt to a mammal having a viral infection.

Another aspect of the invention is an antiviral pharmaceuticalcomposition comprising β-interferon and DHPG or a pharmaceuticallyacceptable salt in a topical formulation.

Another aspect of the invention is an article of manufacture forco-administration comprising two containers, one container containing9-(1,3-dihydroxy-2-propoxymethyl)guanine or a pharmaceuticallyacceptable salt with a pharmaceutically acceptable excipient, andanother container containing natural, recombinant, or modifiedβ-interferon with a pharmaceutically acceptable excipient.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

β-interferon is derived from mammalian cells such as fibroblast cells.As used herein, "β-interferon" includes β-interferon derived both fromnatural sources, including human, bovine, and equine, and by recombinantDNA techniques. It also includes modified forms of β-interferon; e.g.,by glycosylation, methylation, substitution and/or deletion of a limitednumber of amino acids. As used herein, HuIFN-β refers to humanβ-interferon, and rHuIFN-β refers to HuIFN-β produced using recombinanttechniques. IFN-β_(ser-17) refers to β-interferon in which theseventeenth amino acid has been replaced with serine.

Interferon concentrations are commonly expressed as standard "units"which are internationally accepted and documented, and relate to thepotency of a given quantity of interferon to inhibit virus replicationunder standard conditions.

9-(1,3-Dihydroxy-2-propoxymethyl)guanine, sometimes referred to hereinas DHPG, has the following structure: ##STR1##

DHPG may be used as the free base, as a pharmaceutically acceptablesalt, or as a mixture thereof.

The broadest statement of the invention is a composition comprising DHPGor a pharmaceutically acceptable salt thereof and β-interferon.

A preferred form of the invention is a composition comprising DHPG and aform of human β-interferon.

Another preferred embodiment of the invention is the compositioncomprising DHPG with bovine β-interferon.

Another preferred embodiment of the invention is the compositioncomprising DHPG with equine β-interferon.

A more preferred embodiment of the invention is a composition comprisingDHPG and recombinant human β-interferon.

Another preferred embodiment is the composition comprising effectiveamounts of human β-interferon and DHPG in a form suitable for topicalapplication, e.g., as a cream, ointment, or gel.

Another preferred embodiment is the article of manufacture comprising avial containing DHPG or a pharmaceutically acceptable salt thereof witha pharmaceutically acceptable excipient and a vial containingβ-interferon with a pharmaceutically acceptable excipient, in a formsuitable for co-administration, either simultaneously or sequentially,including administration of DHPG and IFN-β at unequal time intervals.

The most preferred embodiment is an ointment for topical applicationcomprising therapeutically effective amounts of recombinant humanβ-interferon_(ser-17) and DHPG.

The process for making DHPG and its pharmaceutically acceptable salts istaught in U.S. Pat. No. 4,355,032, which is incorporated herein byreference. β-interferon may be collected from natural sources by methodswell taught in the art (for example, as by the methods taught in U.S.Pat. No. 4,007,086, incorporated herein by reference), or may beobtained through recombinant DNA techniques. β-interferon may bepurified, e.g., by the process taught by U.S. Pat. No. 4,450,103, whichis incorporated herein by reference, and by other methods taught in theart [see e.g., Tan, Y. H. et al, J. Biol. Chem., 254: 8067-73 (1979),Knight, Jr. E., et al, Science, 207: 525-26 (1979), Okamura H. et al,Biochemistry, 19, 3831-35 (1980)]. The rHuIFN-β_(ser-17) used in theexamples was obtained from Cetus Corp., but may be made by the followingmethod.

IFN-β_(ser-17) is best produced by modifying DNA sequences which codefor IFN-β, the manipulating microorganisms to express the modified DNAas protein. When the first base of codon 17 (thymine) of the sensestrand of the DNA sequence which codes for the mature IFN-β is replacedwith adenine, the cysteine residue at position 17 in the amino acidsequence of IFN-β is replaced by serine. By changing T to other bases,and by changing other bases in codon 17, cysteine may be replaced withother amino acids. The site-specific mutagenesis is induced using asynthetic 17-nucleotide primer having the sequence GCAATTTTCAGAGTCAGwhich is identical to a seventeen nucleotide sequence on the sensestrand of IFN-β in the region of codon 17 except for a single basemismatch at the first base of codon 17. (As used herein,C=deoxycytidine, T=deoxythymidine, A=deoxyadenosine, andG=deoxyguanosine.) The mismatch is at nucleotide 12 in the primer. The17-mer is hydridized to single-stranded M13 phage DNA which carries theantisense strand of the IFN-β gene. The oligonucleotide primer is thenextended on the DNA using DNA polymerase I Klenow fragment (a fragmentof DNA polymerase I lacking the 5'-exonuclease subunit) and theresulting double-strand DNA (dsDNA) is converted to closed circular DNAwith T₄ ligase. Replication of the resulting mutational heteroduplexyields clones from the DNA strand containing the mismatch. Mutant clonesmay be identified and screened by the appearance or disappearance ofspecific restriction sites, antibiotic resistance or sensitivity, or byother methods known in the art. When cysteine is substituted by serine,the substitution of T by A results in the creation of a new Hinflrestriction site in the structural gene. (A restriction site is a pointin a DNA sequence that is recognized and cleaved by a particularrestriction enzyme. A HinfI restriction site is a restriction siterecognized by HinfI endonuclease.) The mutant clone is identified byusing the oligonucleotide primer as a probe in a hybridization screeningof the mutated phage plaques. The primer will have a single mismatchwhen hybridized to the parent but will have a perfect match whenhybridized to the mutated phage DNA. Hybridization conditions can thenbe devised where the oligonucleotide primer will preferentiallyhybridize to the mutated DNA but not to the parent DNA. The newlygenerated Hinfl site also serves as a means of confirming the singlebase mutation in the IFN-β gene.

The M13 phage DNA carrying the mutated gene is isolated and spliced intoan appropriate expression vector, such as plasmid pTrp3, and E. colistrain MM294 is transformed with the vector. Suitable growth media forculturing the transformants and their progeny are known to those skilledin the art. The expressed mutein (protein derived from a mutated gene)of IFN-β is isolated, purified and characterized.

UTILITY AND ADMINISTRATION

The subject composition exhibits potent antiviral activity whenadministered to mammals. For example, the composition of the presentinvention exhibits excellent activity against herpes simplex virus 1 and2 and related viruses such as cytomegalovirus, Epstein-Barr virus andvaricella zoster virus, as well as viral hepatitis such as hepatitis B.

Pharmaceutical compositions, both veterinary and human, containing thesubject composition appropriate for antiviral use are prepared bymethods and contain excipients which are well known in the art. Agenerally recognized compendium of such methods and ingredients isRemington's Pharmaceutical Sciences by E. W. Martin, (Mark Publ. Co.,15th Ed., 1975). Liposomes may also be employed in compositions of theinvention, using methods known in the art [for example, as described inSzoka, F. Jr., et al, Ann. Rev. Biophys. Bioeng. 9:467-508 (1980):Schullery, S. E. et al, Biochemistry 19:3919-23 (1980); Gregoriadis, G.,et al, "Liposomes in Biological Systems:", John Wiley and Sons (1980),and Gregoriadis, G. (Ed.) "Liposome Technology", CRC Press, Inc.(1984).].

When the composition of the invention is administered by methods otherthan topical application, it may be desirable to administer the DHPGcomponent and the β-interferon component at different times, and/or atdifferent frequencies. Also, the components need not be administered bythe same method, e.g., DHPG may be administered orally twice daily whileβ-interferon is administered intramuscularly or topically.

The composition of the invention is effective in mammals, includinge.g., humans, cattle, and horses, and may be administered parenterally(for example, by intravenous, subcutaneous, intraperitoneal,intralesional or intramuscular injection), orally, (DHPG componentonly), topically, rectally or intranasally.

DHPG is administered orally or parenterally at dose levels, calculatedas the free base of DHPG, of about 0.01 to 300 mg/kg, preferably 0.1 to10 mg/kg of mammal body weight, and is used in humans in a unit dosageform, administered one to five times daily in the amount of 1 to 500 mgper unit dose. For oral administration, fine powders or granules maycontain diluting, dispersing and surface active agents, and may bepresented in a draught, in water or in a syrup; in capsules or sachetsin the dry state or in a non-aqueous solution or suspension, whereinsuspending agents may be included; in tablets, wherein binders andlubricants may be included; in a suspension in water or a syrup; or inan aerosol. Where desirable or necessary, flavoring, preserving,suspending, thickening or emulsifying agents may be included. Tabletsand granules are preferred, and these may be coated. The amount of DHPGin the formulation may vary from 0.1 percent weight (%w/w) to 99%w/w ormore of the compound based on the total formulation and about 1%w/w to99.9%w/w excipient. Preferably DHPG is present at a level of 10%-95%w/w.

For parenteral administration of the DHPG component or foradministration as drops, as for eye infections, DHPG may be presented inaqueous solution in a concentration of from about 0.1 to 10% by weightfor DHPG component, more preferably about 0.1 to 0.4%. The solution maycontain pharmaceutically acceptable antioxidants, buffers, and othersuitable additives.

The β-interferon component is administered parenterally in aqueoussolution at dose levels of 10 to 10⁷ U/kg, preferablly 10³ to 10⁵ U/kgof body weight in man, administered once or twice a day for 1 to 7 daysper week. For eye drops, the interferon is present at 10³ -10⁹ U/ml,preferably about 10⁵ -10⁸ U/ml.

Alternatively, for infections of the eye or other external tissues, e.g.mouth, skin, and genitalia, the compositions are preferably applied tothe infected part of the patient's body topically as an ointment, cream,gel, aerosol or powder, preferably in an ointment or cream. Thecompounds may be presented in an ointment, for instance with awater-soluble ointment base, or in a cream, for instance with anoil-in-water cream base, in a concentration of from about 0.01 to 10%for the DHPG component; preferably 0.1 to 7%, most preferably about3%w/v and in a concentration of 10³ -10⁹ U/g, preferably 10⁵ -10⁸ U/gfor the interferon component. Additionally, viral infections may betreated by use of a sustained release drug delivery system such as isdescribed in U.S. Pat. No. 4,217,898.

For aerosol administration, the active ingredient is preferably suppliedin finely divided form or in a solution along with a surfactant and apropellant. Typical percentages of active ingredients are 0.001 to 20%by weight, preferably 0.004 to 1.0%.

Surfactants must, of course, be non-toxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olestearic and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride such as, for example, ethylene glycol, glycerol,erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derivedfrom sorbitol (the sorbitan esters sold under the trademark "Spans") andthe polyoxyethylene and polyoxypropylene derivatives of these esters.Mixed esters, such as mixed or natural glycerides may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the trademarks "Arlacel C" (Sorbitan sesquioleate), "Span 80"(sorbitan monooleate) and "Span 85" (sorbitan trioleate). The surfactantmay constitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquefiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane; andpreferably fluorinated or fluorochlorinated alkanes, such as are soldunder the trademark "Freon." Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

PREPARATION 1 Cloning of the IFN-β gene into M13 Vector

The use of M13 phage vector as a source of single-stranded DNA templatehas been demonstrated by G. F. Temple et al Nature (1982) 296:537-540.Plasmid pβ trp containing the IFN-β gene, under control of E. coli trppromoter, is digested with the restriction enzymes HindIII and XhoII.The M13mp8 (J. Messing, "Third Cleveland Symposium on Macromolecules:Recombinant DNA," Ed. A. Walton, Elsevier Press, 143-153 (1981)replicative form (RF) DNA is digested with restriction enzymes HindIIIand BamHI and mixed with the pβ1 trp DNA which have previously beendigested with HinDIII and XhoII. The mixture is then ligated with T₄ DNAligase and the ligated DNA transformed into competent cells of E. colistrain JM 103 and plated on Xgal indicator plates (J. Messing et al,Nucleic Acids Res (1981) 9:309-321). Plaques containing recombinantphage (white plaques) are picked, inoculated into a fresh culture of JM103 and minipreps of RF molecules prepared from the infected cells (H.D. Birnboim and J. Doly, Nucleic Acid Res. (1979) 7:1513-1523). The RFmolecules are digested with various restriction enzymes to identify theclones containing the IFN-β insert. Single-stranded (ss) phage DNA isprepared from clone M13-β1 to serve as a template for site-specificmutagenesis using a synthetic oligonucleotide.

PREPARATION 2 Site specific mutagenesis

Forty picomoles of the synthetic oligonucleotide GCAATTTTCAGAGTCAG(primer) is treated with T₄ kinase in the presence of 0.1 mM adenosinetriphosphate (ATP). 50 mM hydroxymethylaminomethane hydrochloride(Tris-HCl) pH 8.0, 10 mM MgCl₂, 5 mM dithiothreitol (DTT) and 9 units ofT₄ kinase, in 50 μl at 37° C. for 1 hr. The kinased primer (12 pmole) ishybridized to 5 μg of ss M13-β1 DNA in 50 μl of a mixture containing 50mM NaCl, 10 mM tris-HCl, pH 8.0, 10 mM MgCl₂ and 10 mM β-mercaptoethanolby heating at 67° C. for 5 min and at 42° C. for 25 min. The annealedmixture is then chilled on ice and then added to 50 μl of a reactionmixture containing 0.5 mM each of deoxynucleotide triphosphate (DNTP),80 mM Tris-HCl, pH 7.4, 8 mM MgCl₂, 100 mM NaCl, 9 units of DNApolymerase I Klenow fragment, 0.5 mM ATP and 2 units of T₄ DNA ligase,incubated at 37° C. for 3 hr and at 25° C. for 2 hr. The reaction isthen terminated by phenol extraction and ethanol precipitation. The DNAis dissolved in 10 mM Tris-HCl pH 8.0, 10 mM ethylenediaminetetraaceticacid (EDTA), 50% sucrose and 0.05% bromophenylblue and electrophoresedon 0.8% agarose gel in the presence of 2 μg/ml of ethidium bromide. TheDNA bands corresponding to the RF forms of M13-β1 are eluted from gelslices by the perchlorate method (R. W. Davis, et al, "AdvancedBacterial Genetics," Cold Spring Harbor Laboratory, N.Y., p. 178-179(1980)). The eluted DNA is used to transform competent JM 103 cells,grown overnight and single strand (ss) DNA isolated from the culturesupernatant. This ssDNA is used as a template in a second cycle ofprimer extension, the gel purified RF forms of the DNA are transformedinto competent JM 103 cells, plated onto agar plates and incubatedovernight to obtain phage plaques.

PREPARATION 3 Screening and identification of mutagenized plaques

Plates containing mutated M13-β1 plaques as well as two platescontaining unmutated M13-β1 phage plaques are chilled to 4° C., andplaques from each plate transferred onto two nitrocellulose filtercircles by layering a dry filter on the agar plate for 5 min for thefirst filter and 15 min for the second filter. The filters are thenplaced on thick filter papers soaked in 0.2N NaOh, 1.5M NaCl and 0.2%Triton X-100 for 5 min. and neutralized by layering onto filter paperssoaked with 0.5M Tris-HCl, pH 7.5 and 1.5M NaCl for another 5 min. Thefilters are washed in a similar fashion twice on filters soaked in 2xSSC(standard saline citrate), dried and then baked in a vacuum oven at 80°C. for 2 hr. The duplicate filters are prehybridized at 55° C. for 4 hr.with 10 ml per filter of DNA hybridization buffer (5xSSC) pH 7.04xDenhardt's solution (polyvinyl-pyrrolidine, ficoll and bovine serumalbumin, 1x=0.02% of each), 0.1% sodium dodecyl sulfate (SDS), 50 mMsodium phosphate buffer pH 7.0 and 100 μg/ml of denatured salmon spermDNA. A ³² P-labeled probe is prepared by kinasing the oligonucleotideprimer with ³² P-labeled ATP. The filters are hybridized to 3.5×10⁵cpm/ml of ³² P-labeled primer in 5 ml per filter of DNA hybridizationbuffer at 55° C. for 24 hr. The filters are washed at 55° C. for 30 min.each in washing buffers containing 0.1% SDS and decreasing amounts ofSSC. The filters are washed initially with buffer containing 2xSSC andthe control filters containing unmutated M13-β1 plaques are checked forthe presence of any radioactivity. The concentration of SSC is loweredstepwise and the filters washed until no detectable radioactivityremains on the control filters with the unmutated M13-β1 plaques. Thefilters are air dried and autoradiographed at -70° C. for 2-3 days.

PREPARATION 4 Expression of mutated IFN-β in E. coli

RF DNA from M13-SY2501 is digested with restriction enzymes HindIII andXhoII and the 520 bp insert fragment purified on a 1% agarose gel. Theplasmid pTrp3 containing the E. coli trp promoter is digested with theenzymes HindIII and BamHI, mixed with the purified M13-SY2501 DNAfragment and ligated in the presence of T₄ DNA ligase. The ligated DNAis transformed into E. coli strain MM294. Ampicillin resistanttransformants are screened for sensitivity to the drug tetracycline.Plasmid DNA from five ampicillin resistant, tetracycline-sensitiveclones are digested with Hinfl to screen for the presence of theM13-SY2501 insert.

The plasmid designated as clone pSY2501 is available from theAgricultural Research Culture Collection (NRRL), FermentationLaboratory, Northern Regional Research Center, Science and EducationAdministration, U.S. Department of Agriculture, 1815 North UniversityStreet, Peoria, Ill. 60604 and is assigned accession numbers CMCC No.1533 and NRRL No. B-15356.

Cultures of pSY2501 and pβltrp are grown up to an optical density(DD₆₀₀) of 1.0. Cell free extracts are prepared and the amount ofIFN-βantiviral activity assayed on GM2767 cells in a microtiter assay.

PREPARATION 5 Purification of IFN-β_(ser17)

IFN-β_(ser17) is recovered from E. coli which have been transformed toproduce IFN-β_(ser17). The E. coli are grown in the following growthmedium to an OD of 10-11 at 680 nm (dry wt 8.4 g/l).

    ______________________________________                                        Ingredient            Concentration                                           ______________________________________                                        NH.sub.4 Cl           20 mM                                                   K.sub.2 SO.sub.4      16.1 mM                                                 KH.sub.2 PO.sub.4     7.8 mM                                                  Na.sub.2 HPO.sub.4    12.2 mM                                                 MgSO.sub.4.7H.sub.2 O 3mM                                                     Na.sub.2 citrate.2H.sub.2 O                                                                         1.5 mM                                                  MnSO.sub.4.4H.sub.2 O 30 μM                                                ZnSO.sub.4.7H.sub.2 O 30 μM                                                CuSO.sub.4.5H.sub.2 O 3 μM                                                 L-tryptophan          70 mg/l                                                 FeSO.sub.4.7H.sub.2 O 72 μM                                                thiamine HCl          20 mg/l                                                 glucose               40 G/L                                                  pH controlled with NH.sub.4 OH                                                ______________________________________                                    

A 9.9 l (9.9 kg) harvest of the transformed E. coli is cooled to 20° C.and concentrated by passing the harvest through a cross-flow filter atan average pressure drop of 110 kPa and steadystate filtrate flow rateof 260 ml/min until the filtrate weight is 8.8 kg. The concentrate(approximately one liter) is drained into a vessel and cooled to 15° C.The cells in the concentrate are then disrupted by passing theconcentrate through a Mason-Gaulin homogenizer at 5° C. 69,000 kPa. Thehomogenizer is washed with one liter phosphate buffered saline, pH 7.4(PBS), and the wash is added to the disruptate to give a final volume oftwo liters. This volume is continuously centrifuged at 12000xg at a 50ml/min flow rate. The solid is separated from the supernatant andresuspended in four liters of PBS containing 2% by wt. SDS. Thissuspension is stirred at room temperature for 15 min after which thereshould be no visible suspended material. The solution is then extractedwith a 2-butanol at a 1:1 2-butanol:solution volume ratio. Theextraction is carried out in a liquid-liquid phase separator using aflow rate of 200 ml/min. The organic phase is then separated andevaporated to dryness to yield 21.3 g of protein. This may then beresuspended in distilled water at a 1:10 volume ratio.

EXAMPLE 1

Human embryonic tonsil (HET) cells were plated in 24 well dishes at 10⁵cells/well and incubated 24 hr until confluent monolayers were obtained.Cells were then treated for 24 hr with interferon α, β, or γ asindicated, or with media without interferon. The cells were subsequentlywashed and infected with herpes simplex virus type 2 (HSV-2) at amultiplicity of infection (MOI) of 5 plaque forming units (PFU) percell. Virus was absorbed for 1 hr at 37° C., and the cells were thenwashed and the interferons and/or DHPG were added as indicated totriplicate wells. Cell-virus cultures were then incubated for 1 day, andthe virus yield was subsequently determined by plaque assay in Verocells.

As shown in Table 1, 0.03 μM DHPG alone reduced the yield of infectiousHSV-2 by 1.9 fold. Treatment with interferons α₂ or γ alone at 200 U/mldid not significantly affect virus yield, while treatment withinterferon α₁ or β reduced virus yield by 1.4 and 2.7 fold respectively.However, the most striking reduction in virus yield was obtained by thecombination of DHPG and β-interferon, resulting in a 1750-fold reductionin virus yield.

                  TABLE 1                                                         ______________________________________                                        Inhibition of Growth of HSV-2 by Interferons Alone                            or in Combination with DHPG                                                   Interferon                                                                    type:       Ratio of Virus Yield (Untreated/Treated)                          DHPG  (IU/ml)   none    α.sub.1                                                                       α.sub.2                                                                       β-ser.sub.17                                                                     γ                           ______________________________________                                        0     0         ≡ 1                                                                             --    --    --      --                                0.03  0         1.9     --    --    --      --                                0     200       --      1.4   0.9   2.7     1.0                               0.03  200       --      250.  100   1750    10                                ______________________________________                                    

This unexpected and surprising synergistic reduction of virus yield byDHPG/β-interferon combination was significantly greater than thereduction obtained by combination of DHPG with either α or γinterferons. As shown in Table 2, the relative increase in foldreduction of virus by β-interferon/DHPG combination vs α₁, α₂, orγ-interferon/DHPG combination ranged from 7 to 175 fold. Thus theβ-interferon/DHPG combination was significantly more potent ininhibiting viral growth than were the α or γ-interferon/DHPGcombinations.

                  TABLE 2                                                         ______________________________________                                        Increase in Synergy of β-interferon/DHPG Combination vs.                 α.sub.1, α.sub.2, or γ-interferon/DHPG                      ______________________________________                                        Combinations                                                                  Increase in Fold Reduction of Virus Yield by β-IFN                        ##STR2##                                                                                  ##STR3##                                                                                     ##STR4##                                          7           18             175                                                ______________________________________                                    

EXAMPLE 2

HET cells were treated with interferons and/or DHPG as described inExample 1, except that additional cultures were treated similarly with9-(2-hydroxy-1-ethoxymethyl)guanine (ACV, acyclovir) with or without thevarious interferons. The results show that the antiherpetic synergybetween DHPG and β-interferon was significantly greater (50-120 fold)than the combination of ACV and β-interferon. Also, the increasedantiherpetic activity of the DHPG/β-interferon combination vs theACV/β-interferon combination was additionally much greater than theincreased antiherpetic activity of DHPG/α-interferon over that ofACV/α-interferon, as well as that of DHPG/γ-interferon over that ofACV/γ-interferon (Table 3).

                  TABLE 3                                                         ______________________________________                                        Increased Inhibition of HSV-2 Growth by DHPG-Interferon                       Treatment as Compared to ACV-Interferon Treatment                             rHuIFN    DHPG or ACV Ratio of HSV-2 Yield                                    Type  IU/ml   (μM)     (ACV + IFN/DHPG + IFN)                              ______________________________________                                        none  --      0.03        1.0                                                       --      0.1         2.0                                                 α.sub.1                                                                       200     0.03        50                                                        100     0.07        20                                                  α.sub.2                                                                       200     0.03        20                                                        100     0.07        10                                                  β-ser.sub.17                                                                   200     0.03        120                                                       100     0.07        50                                                  γ                                                                             200     0.03        3                                                   ______________________________________                                    

Thus the above results show that DHPG is strikingly synergistic withβ-interferon in inhibiting growth of herpes simplex virus (1750 foldreduction in virus yield vs 2-3 fold reduction by either DHPG orβ-interferon alone), and that the combination of DHPG with β-interferonis surprisingly more effective (7-175 fold more effective) than is thecomparable combination of DHPG with either α or γ interferons (Example1). Furthermore, our results show that the antiherpetic synergismobtained by DHPG in combination with β-interferon is substantially moreeffective (up to 120 times) than is a comparable combination of ACV withβ-interferon (Example 2).

EXAMPLE 3

The following example illustrates the preparation of representativepharmaceutical formulations containing DHPG and human β-interferon.

    ______________________________________                                        A. Topical Formulation                                                        ______________________________________                                        DHPG                     5.0                                                  β-Interferon        10..sup.8                                                                            U                                             Span 60                  2      g                                             Tween 60                 2      g                                             Mineral oil              5      g                                             Petrolatum               10     g                                             Methyl paraben           0.15   g                                             Propyl paraben           0.05   g                                             BHA (butylated hydroxy anisole)                                                                        0.01   g                                             Water qs                 100    ml                                            ______________________________________                                    

All of the above ingredients, except water and interferon, are combinedand heated at 60° C. with stirring. A sufficient quantity of water at60° C. is then added with vigorous stirring to provide 90-99 g of thecream which is then cooled. The interferon which may be mixed withstabilizers such as albumin, dextrose, and pH buffering agents, isdissolved in 1-10 ml water to bring the total cream formulation to 100g, and is then added with sufficient stirring, and the cream formulationis then cooled to room temperature or lower.

The following formulation is useful for intraperitoneal, subcutaneous,intralesional and intramuscular injection.

    ______________________________________                                        B. IP, SC, IL and IM Formulation                                              ______________________________________                                        DHPG                  300.0    mg                                             β-Interferon     2 × 10.0.sup.7                                                                   U                                              Propylene glycol      20       g                                              Polyethylene glycol   20       g                                              Tween 80              1        g                                              0.9% Saline solution qs                                                                             100      ml                                             ______________________________________                                    

The DHPG and β-interferon are dissolved in propylene glycol,polyethylene glycol 400 and Tween 80. A sufficient quantity of 0.9%saline solution is then added with stirring to provide 100 ml of the IP,SC, IL or IM solution which is filtered through a 0.2 micron membranefilter and packaged under sterile conditions.

The following formulation is useful for intravenous injection.

    ______________________________________                                        C. I.V. Formulation                                                           ______________________________________                                        DHPG                   300    mg                                              β-Interferon      10.sup.6                                                                             U                                               Polysorbate 80         0.1    g                                               Propylene glycol or    3.0    g                                               polyethylene glycol 400                                                       Water qs               100    ml                                              ______________________________________                                    

The active compounds are added to a solution of polysorbate 80 andpropylene glycol or polyethylene glycol 400 in 20 ml of water and mixed.The resulting solution is diluted with water to 100 ml and filteredthrough the appropriate 0.2 micron membrane filter.

    ______________________________________                                        D. Tablet Formulation                                                                          Parts by weight                                              ______________________________________                                        DHPG               200                                                        Magnesium stearate  3                                                         Starch              30                                                        Lactose            116                                                        PVP (polyvinylpyrrolidone)                                                                        3                                                         ______________________________________                                    

The above ingredients are combined and granulated using methanol as thesolvent. The formulation is then dried and formed into tablets(containing 200 mg of active DHPG compound) with an appropriatetableting machine. (β-interferon is co-administered non-orally, e.g.,topically, intramuscularly, intravenously, subcutaneously,intralesionally).

What is claimed is:
 1. A composition comprising an effective amount ofhuman β-interferon(ser 17) and 9-(1,3-dihydroxy-2-propoxymethyl)guanineor a pharmaceutically acceptable salt thereof.
 2. An antiviralpharmaceutical composition which comprises an effective amount of thecomposition of claim 1 and a pharmaceutically acceptable carrier.
 3. Theantiviral pharmaceutical composition of claim 2, which is suitable fortopical application.
 4. A method of treating viral infections in amammal having a viral infection which comprises administering aneffective amount of the composition of claim
 1. 5. An article ofmanufacture for treating a viral infection with an effective amount ofhuman β-interferon(ser 17) and 9-(1,3-dihydroxy-2-propoxymethyl)guaninein a synergistic ratio, which article of manufacture comprises:a firstcontainer containing human β-interferon(ser 17) and a firstpharmaceutically acceptable excipient suitable for parenteraladministration; and a second container containing9-(1,3-dihydroxy-2-propoxymethyl)guanine or a pharmaceuticallyacceptable salt thereof and a second pharmaceutically acceptableexcipient.
 6. The article of manufacture of claim 5 wherein said secondpharmaceutically acceptable excipient is suitable for oraladministration.